Cloning and nucleotide sequence of the gyrB gene of Vibrio parahaemolyticus and its application in detection of this pathogen in shrimp.
Because biochemical testing and 16S rRNA sequence analysis have proven inadequate for the differentiation of Vibrio parahaemolyticus from closely related species, we employed the gyrase B gene (gyrB) as a molecular diagnostic probe. The gyrB genes of V. parahaemolyticus and closely related Vibrio alginolyticus were cloned and sequenced. Oligonucleotide PCR primers were designed for the amplification of a 285-bp fragment from within gyrB specific for V. parahaemolyticus. These primers recognized 117 of 117 reference and wild-type V. parahaemolyticus strains, whereas amplification did not occur when 90 strains of 37 other Vibrio species or 60 strains representing 34 different nonvibrio species were tested. In 100-microliter PCR mixtures, the lower detection limits were 5 CFU for live cells and 4 pg for purified DNA. The possible application of gyrB primers for the routine identification of V. parahaemolyticus in food was examined. We developed and tested a procedure for the specific detection of the target organism in shrimp consisting of an 18-h preenrichment followed by PCR amplification of the 285-bp V. parahaemolyticus-specific fragment. This method enabled us to detect an initial inoculum of 1.5 CFU of V. parahaemolyticus cells per g of shrimp homogenate. By this approach, we were able to detect V. parahaemolyticus in all of 27 shrimp samples artificially inoculated with this bacterium. We present here a rapid, reliable, and sensitive protocol for the detection of V. parahaemolyticus in shrimp.
Venkateswaran
K
K
Nippon Suisan Kaisha, Ltd., Central Research Laboratory, Tokyo, Japan. kjvenkat@csd.uwm.edu
Dohmoto
N
N
Harayama
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II / Q000235:genetics*; D033364:Decapoda / Q000382:microbiology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014736:Vibrio parahaemolyticus / Q000235:genetics* / Q000302:isolation & purification*
D027081:DNA Gyrase; D004250:DNA Topoisomerases, Type II